© F e r r a t a S t o r t i F o u n d a t i o nphic alleles of the C3 gene and of the complement receptor 1 (CR1) gene. Methods PatientsSeventy-two patients with hemolytic PNH who had received eculizumab treatment were analyzed after a median follow-up of 52 months (range, 11-98 months). Patients with evidence of bone marrow failure (reticulocytes ≤60x10 9 /L, platelets ≤50x10 9 /L, neutrophils ≤1x10 9 /L) were not included in this series because this condition may affect the clinical response to eculizumab regardless of how well hemolysis is controlled. Peripheral blood samples were collected for clinical testing, flow cytometry (GPI-molecules and C3-binding) 7 and genotyping after the patients had signed an informed consent form approved by the respective Institutional Review Board. As a criterion of response to eculizumab we used the one that is most stringent and objective: namely RBC transfusion at any time after the first 6 months on eculizumab treatment. Of the 72 patients 60 were transfusiondependent before eculizumab: of these 60, those who on eculizumab received no further transfusion during follow-up have been classified as good responders; those who on eculizumab received one or more RBC transfusion at any time during follow-up have been classified as suboptimal responders. Twelve patients started eculizumab before having received any RBC transfusions. Of these, the ten patients who on eculizumab remained transfusion-independent have been classified as good responders because their hemoglobin level increased, on average, by 2.2 g/dL; the remaining two patients required RBC transfusions on eculizumab and have been classified as sub-optimal responders. Only one patient, who had hemolytic PNH at the time of starting eculizumab and who had already been classified as a sub-optimal responder, became aplastic during follow-up. GenotypingThe polymorphism rs2230199 C>G of the C3 gene was investigated by a newly designed tetra-primer amplification refractory mutation system-polymerase chain reaction method.11 Three polymorphisms of the CR1 gene were genotyped by restriction fragment length (RFLP) analysis:12 HindIII RFLP (intron 27); His1208Arg (exon 22); Pro1827Arg (exon 33). Kinetics of C3 binding in vitroRBC and sera were promptly separated from peripheral blood freshly collected from PNH patients. The RBC were then incubated with sera containing eculizumab as previously described. 13,14 Briefly, a 2% suspension of RBC from PNH patients was incubated at 37°C with a pool of ABO-compatible sera from patients on eculizumab, and the complement alternative pathway was activated by mild acidification (HCl 0.016 M). At serial time points (15,30, 60, and 120 min) after complement activation the fraction of GPI-negative RBC with newly bound C3 fragments was measured by flow cytometry (AccuriC6, Becton Dickinson, NJ, USA) after staining with anti-CD59 Alexa647 (Mem43, Serotec, UK) and anti-C3d-neoantigen (A250, Quidel, CA, USA); secondary staining was performed with phycoerythrin-labeled polyclonal rabbit-an...

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